1. Field of the Invention
The invention relates to compositions and methods for the assessment of mucin gene expression. The invention also relates to compositions and methods for the identification of compounds useful in the treatment of various medical conditions caused by mucin overproduction.
2. Description of the Related Art
Mucins are a family of high molecular weight glycoproteins secreted from epithelial cells at many body surfaces, including the eyes, pancreatic ducts, gallbladder, prostate and respiratory, gastrointestinal and reproductive tracts. Mucins are a major component of mucus, and are responsible for the viscoelastic properties of mucus, and serve a role in protecting and lubricating the epithelial surfaces. At least twelve mucin genes have been identified in humans.
In the airways, mucin proteins form a protective barrier on the airway epithelial cells, and interact with cilia to trap and clear pathogens (e.g., microorganisms), particulate matter, irritants and pollutants (e.g., tobacco smoke and sulfur dioxide). Mucus secretions in the airway are produced from two different secretory cell populations, the surface epithelial goblet cells and the mucous cells in the submucosal glands. At least eight mucin genes are expressed (at the mRNA level) in the upper and lower respiratory tracts. Of these, only the MUC5AC and MUC5B polypeptides have been conclusively demonstrated to be major components of human airway secretions (Hovenberg et al., Biochem. J., 318(Pt. 1, Vol. 17):319-324 [1996]; Hovenberg at al., Glycoconjugate Jour., 13(5):839-847 [1996]; Thornton et at., J. Biol. Chem., 272(14):9561-9566 [1997]; and Wickstrom et al., Biochem. Jour., 334(Pt. 3, Vol. 14):685-693 [1998]). MUC5B is also expressed in other tissues, including, for example, pancreas and gall bladder.
Diseases of Mucin Overproduction
Mucin production is upregulated in response to mucosal irritation. Most notably, bacterial infection of the airway epithelium is often accompanied by mucin overproduction. Some airway diseases are also characterized by mucus hypersecretion. Hypersecretion of mucus can overwhelm the ability of the cilia to function properly, and can result in various pathologies, such as airway mucus plugging and airflow obstruction. Mucus hypersecretion also contributes to chronic infection by shielding bacteria from endogenous and exogenous antibacterial agents. Mucus plugging and bacterial infections create a non-healing injury and can result in chronic influx of inflammatory cells which destroy gas exchange tissue. When severe, these effects result in respiratory function decline, and can be fatal.
Diseases which are characterized by mucin (and mucus) hypersecretion also frequently demonstrate goblet cell hyperplasia and submucosal gland hypertrophy. Such diseases include, for example, chronic bronchitis, bronchial pneumonia, cystic fibrosis, chronic asthma, emphysema, usual interstitial pneumonitis and other diseases (Basbaum et al., Am. Rev. Respir. Dis., 144(3 Pt 2):S38-41 [1991]; Yanagihara et al., Am. J. Respir. Cell. Mol. Biol., 24(1):66-73 [2001]; Rogers et al., Eur. Respir. J., 7(9):1690-706 [1994]; and Kaliner et al., American Review of Respiratory Disease 134(3):612-21[1986]).
MUC5B mRNA and Genomic Structure
In order to better understand the molecular mechanism of mucin gene expression regulation in normal and disease states, it is necessary to elucidate the genomic structure of the mucin gene. MUC5B and three other mucin genes, MUC6, MUC2, and MUC5AC, have all been mapped to 11p15.5 on a single band of 400 kilobases, and their order has been determined to be: telomere-MUC6-MUC2-MUC5AC-MUC5B-centromere. The MUC5B genomic structure (i.e., exon identification, intron/exon boundaries and transcriptional start sites) and cDNA sequence are also partially known, albeit with some discrepancies in the published literature (Pigny et al., Genomics 38(3):340-352 [1996]; Desseyn et al., Jour. Biol. Chem., 273(46):30157-30164 [1998]; Desseyn et al., Jour. Biol. Chem., 272(27):16873-16883 [1997]; Desseyn et al., Jour. Biol. Chem., 272(6):3168-3178 [1997]; Offner et al., Biochem. Biophy. Res. Comm., 251(1):350-355 [1998]; and Keates et al., Biochem. J., 324(Pt 1):295-303 [1997]).
The MUC5B gene is large and complex. The MUC5B exons and introns encompass approximately 39076 basepairs of genomic sequence, and the gene""s cDNA is approximately 17079 basepairs in length. The gene is characterized by an unusually large central exon of 10,713 basepairs and 3,571 amino acids. The central exon contains multiple repeated motifs, including characteristic cysteine-rich subdomains, which are also found in other mucin genes. In addition to the large central exon, there are approximately 30 smaller exons upstream and another approximately 17 exons downstream of the central exon. In total, the MUC5B message is predicted to encode a 5683 amino acid polypeptide having a molecular weight of 590 kDa. However, as the mucin proteins are extensively glycosylated, the observed molecular weight is expected to be much greater. Conflicting descriptions of the gene""s transcription start sites and identity of the first exon have been reported.
There exist published reports of the isolation and analysis of limited portions of the MUC5B 5xe2x80x2 promoter region. Van Seuningen et al. (Biochem. J., 348(Pt. 3):675-686 [2000]) describe an analysis of the MUC5B promoter region, which encompasses approximately 956 basepairs of genomic nucleotide sequence upstream of the transcription start site. Perrais et al. (J. Biol. Chem., 276(18):15386-15396 [2001]) describe an analysis of the MUC5B promoter region, which includes approximately 2044 basepairs of genomic nucleotide sequence upstream of the transcription start site. GenBank Accession Number AJ012453 describes approximately 2954 basepairs of MUC5B genomic sequence 5xe2x80x2 of the transcriptional start site.
There is a need to identify compounds capable of inhibiting the production of mucin proteins, and specifically, MUC5B protein. There is a need to provide therapies for reducing mucus (e.g., MUC5B) production in individuals suffering from airway diseases characterized by mucus hypersecretion, such as cystic fibrosis, chronic bronchitis, bronchial pneumonia and asthma. The object of the present invention is to provide novel compositions and methods that find use in the analysis of MUC5B gene expression. These compositions incorporate previously unreported MUC5B genomic sequences derived from the MUC5B gene 5xe2x80x2 promoter region, and the methods of the invention use these sequences. These novel compositions further comprise reporter genes in operable combination with the novel MUC5B gene 5xe2x80x2 promoter regions of the present invention. It is also an object of the present invention to provide methods for drug screening using the novel MUC5B promoter reporter constructs to identify compounds having the ability to downregulate MUC5B gene expression. The invention also provides transgenic animals suitable for use in screening assays to identify compounds capable of inhibiting mucin production. Compounds thus identified find use in the treatment of diseases characterized by mucin hypersecretion.
The present invention provides novel isolated nucleic acid molecules comprising promoter sequences regulating the transcription of the human MUC5B gene. These novel sequences are provided in SEQ ID NO: 31 and SEQ ID NO: 32. In a related embodiment, the invention also provides nucleic acid molecules wherein the promoter sequences of SEQ ID NO: 31 or SEQ ID NO: 32 are operably linked to a heterologous gene (i.e., a gene that is not naturally linked to the promoter sequences of SEQ ID NO: 31 or SEQ ID NO: 32).
In one embodiment, the combination of promoter sequence and heterologous gene reside within a vector. In some embodiments, the heterologous gene contained on the vector is a reporter gene. The heterologous gene can encode various polypeptides, including luciferase, green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT), xcex2-glucuronidase (GUS), secreted alkaline phosphatase (SEAP) and xcex2-galactosidase (xcex2-gal).
It is intended that host cells harboring the nucleic acid molecules and various vectors of the present invention are also within the scope of the invention. The nature of the host cell is not particularly limited. In some embodiments, host cells harboring the nucleic acid molecule comprising either promoter sequences of SEQ ID NO: 31 or SEQ ID NO: 32 operably linked to a heterologous gene are provided by the present invention. Furthermore, host cells harboring a vector carrying either of these promoter sequences operably linked to a heterologous gene are also provided by the invention. In related embodiments, host cells harboring a vector carrying either of these promoter sequences operably linked to a reporter gene are provided by the invention. In some embodiments, the host cell is a eukaryotic cell. In other embodiments, the host cell is a cell of human origin. In some preferred embodiments, the host cell is a cell of tracheobronchial epithelial (TBE) origin. When cells are of TBE origin, they may be primary TBE cells or established HBE1 cells. In one embodiment, when the host cells are eukaryotic cells, the host cell can be present in a non-human mammal, in which case the non-human mammal is a transgenic animal. It is intended that transgenic animals comprising the nucleic acid molecules, vectors and host cells of the invention are within the scope of the invention.
The present invention provides a variety of cell culture conditions and culture methods for the cultivation of the host cells of the invention. In its broadest sense, the invention provides a method for culturing a host cell in a culture medium under conditions allowing the expression of a heterologous gene product that is under the transcriptional control of MUC5B promoter sequences SEQ ID NO: 31 or SEQ ID NO: 32. In one embodiment of these cell culture methods, the host cell is of tracheobronchial epithelial (TBE) origin. In other embodiments, the host cell of TBE origin is cultured biphasically in an air-liquid interface. In still other methods for culturing host cells of the invention, the host cell of TBE origin is cultured on a substrate comprising collagen gel. In still other culture methods, the host cells are cultured in the presence of retinoic acid.
In another embodiment, the present invention provides non-human transgenic mammals comprising eukaryotic host cells harboring the promoter sequences of SEQ ID NO: 31 or SEQ ID NO: 32 operably linked to a heterologous gene.
The present invention provides a wide variety of methods for the assessment of MUC5B promoter activity, and related screening methods to identify compounds having the ability to inhibit human MUC5B promoter activity. In one embodiment, a method for the assessment of MUC5B gene promoter activity entails delivering a reporter construct driven by MUC5B promoter sequences SEQ ID NO: 31 or SEQ ID NO: 32 operably linked to a reporter gene to a host cell, and assessing the expression of said marker gene product encoded by the reporter gene. In this method, expression of the marker gene product is indicative of MUC5B gene promoter activity.
In a related embodiment, the method above further comprises measuring the quantity of the marker gene product, where the quantity of the marker gene product is proportionate to MUC5B gene promoter activity.
In another embodiment, the present invention provides a method for identifying a compound capable of modulating MUC5B gene promoter activity, where the method has the steps of providing a first and a second sample of a host cell, where the host cell harbors a reporter construct driven by a MUC5B nucleotide sequence of SEQ ID NO: 31 or SEQ ID NO: 32, operably linked to a reporter gene encoding a marker gene product; contacting the first sample of host cells with a test compound; assessing the expression of the marker gene product in the first and second samples; and identifying the compound as capable of modulating MUC5B gene promoter activity if the expression of the marker gene product is significantly different in the first and second samples.
In a related embodiment of the method above, the quantity of the marker gene product is measured, where the quantity is proportionate to MUC5B gene promoter activity. Also in a related embodiment of the method above, the modulation is inhibition.
The present invention also provides a method for identifying a compound capable of modulating MUC5B gene promoter activity. In one embodiment, this method comprises the steps of providing a host cell harboring a reporter construct driven by a MUC5B nucleotide sequence of SEQ ID NO: 31 or SEQ ID NO: 32, operably linked to a reporter gene encoding a marker gene product; contacting the host cell with a test compound; measuring the activity of the reporter gene construct; and identifying a compound as capable of modulating MUC5B gene promoter activity, if the activity of the reporter gene construct is significantly different from activity measured prior to contact with the test compound. In one embodiment of this method, the modulation is inhibition.
The present invention provides methods for producing a non-human transgenic animal. In one embodiment, the method comprises the steps of introducing a vector comprising a reporter gene under control of a MUC5B promoter sequence comprising a nucleotide sequence of SEQ ID NO: 31 or SEQ ID NO: 32 into an embryonic stem cell of a non-human transgenic animal to produce a transgenic embryonic stem cell; introducing the transgenic embryonic stem cell into a female mouse under conditions such that the mouse delivers progeny of the transgenic embryonic stem cell; and identifying at least one offspring of the progeny comprising the vector.
In another embodiment of this method, the non-human transgenic animal selectively expresses the reporter gene in a cell of tracheobronchial epithelial (TBE) origin. In another embodiment, the transgenic animal is a mouse.
The present invention provides methods for screening compounds for the ability to modulate MUC5B gene promoter activity. This method comprises the steps of administering a test compound to a non-human transgenic animal produced by the method above, and monitoring MUC5B gene promoter activity. In one embodiment of this method, the modulation is inhibition.
The present invention also provides a method for the specific expression of a nucleic acid of interest in cells of tracheobronchial epithelial (TBE) origin of a mammal, comprising delivering a vector comprising the nucleic acid of interest under control of a MUC5B promoter sequence with a sequence of SEQ ID NO: 31 or SEQ ID NO: 32 to the mammal.